Perinatal hypophosphatasia: radiology, pathology and molecular biology studies in a family harboring a splicing mutation (648+1A) and a novel missense mutation (N400S) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene.

We report on a postmortem diagnosis of perinatal lethal hypophosphatasia, an inborn error of metabolism characterized by a liver/bone/kidney alkaline phosphatase (ALP)-related defective bone mineralization due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. Radiological and pathological studies identified a perinatal lethal hypophosphatasia showing a generalized bone mineralization defect including asymmetry of the cervical vertebral arches in a 22 +4 weeks' gestation fetus. Both parents revealed low serum ALP activities supporting the diagnosis. Sequencing analysis of the TNSALP gene showed two heterozygous mutations, 648+1A, a mutation affecting the donor splice site in exon 6, and N400S, a novel missense mutation in exon 11, located near the active site and very close to histidins 364 and 437, two crucial residues of the active site. Sequencing of exons 6 and 11 in the parents showed that 648+1A was from maternal origin and N400S from paternal origin. DNA-based prenatal testing in the subsequent pregnancy following a chorionic villous sampling performed at 10 weeks of gestation showed no mutation and a healthy infant was born at term.     

Characterization of eleven novel mutations (M45L, R119H, 544delG, G145V, H154Y, C184Y, D289V, 862+5A, 1172delC, R411X, E459K) in the tissue-nonspecific alkaline phosphatase (TNSALP) gene in patients with severe hypophosphatasia. Mutations in brief no. 217. Online

Hypophosphatasia is a rare inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/ bone/kidney tissue alkaline phosphatase (L/B/K ALP) activity. We report the characterization of tissue-nonspecific alkaline phosphatase (TNSALP) gene mutations in a series of 9 families affected by severe hypophosphatasia. Fourteen distinct mutations were found, 3 of which were previously reported in the North American or Japanese populations. Seven of the 11 new mutations were missense mutations (M45L, R119H, G145V, C184Y and H154Y, D289V, E459K), the four others were 2 single nucleotide deletions (544delG and 1172delC), a mutation affecting donor splice site (862 + 5A) and a nonsense mutation (R411X).     

Clinical spectrum and molecular basis of recessive congenital methemoglobinemia in India

We report the clinical features and molecular characterization of 23 patients with cyanosis due to NADH‐cytochrome b5 reductase (NADH‐CYB5R) deficiency from India. The patients with type I recessive congenital methemoglobinemia (RCM) presented with mild to severe cyanosis only whereas patients with type II RCM had cyanosis associated with severe neurological impairment. Thirteen mutations were identified which included 11 missense mutations causing single amino acid changes (p.Arg49Trp, p.Arg58Gln, p.Pro145Ser, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, p.Ala179Thr, p.Thr238Met, and p.Val253Met), one stop codon mutation (p.Trp236X) and one splice‐site mutation (p.Gly76Ser). Seven of these mutations (p.Arg50Trp, p.Gly155Glu, p.Arg160Pro, p.Met177Ile, p.Met177Val, p.Ile178Thr, and p.Thr238Met) were novel. Two mutations (p.Gly76Ser and p.Trp236X) were identified for the first time in the homozygous state globally causing type II RCM. We used the three‐dimensional (3D) structure of human erythrocyte NADH‐CYB5R to evaluate the protein structural context of the affected residues. Our data provides a rationale for the observed enzyme deficiency and contributes to a better understanding of the genotype–phenotype correlation in NADH‐CYB5R deficiency.     

Perinatal hypophosphatasia: Radiology,pathology and molecular biology studies in a family harboring a splicing mutation (648+1A) and a novel missense mutation (N400S) in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene

We report on a postmortem diagnosis of perinatal lethal hypophosphatasia, an inborn error of metabolism characterized by a liver/bone/kidney alkaline phosphatase (ALP)‐related defective bone mineralization due to mutations in the tissue‐nonspecific alkaline phosphatase (TNSALP) gene. Radiological and pathological studies identified a perinatal lethal hypophosphatasia showing a generalized bone mineralization defect including asymmetry of the cervical vertebral arches in a 22 +4 weeks' gestation fetus. Both parents revealed low serum ALP activities supporting the diagnosis. Sequencing analysis of the TNSALP gene showed two heterozygous mutations, 648+1A, a mutation affecting the donor splice site in exon 6, and N400S, a novel missense mutation in exon 11, located near the active site and very close to histidins 364 and 437, two crucial residues of the active site. Sequencing of exons 6 and 11 in the parents showed that 648+1A was from maternal origin and N400S from paternal origin. DNA‐based prenatal testing in the subsequent pregnancy following a chorionic villous sampling performed at 10 weeks of gestation showed no mutation and a healthy infant was born at term. © 2001 Wiley‐Liss, Inc.     

Matrix vesicles in osteomalacic hypophosphatasia bone contain apatite-like mineral crystals.

Hypophosphatasia, a heritable disease characterized by deficient activity of the tissue nonspecific isoenzyme of alkaline phosphatase (TNSALP), results in rickets and osteomalacia. Although identification of TNSALP gene defects in hypophosphatasia establishes a role of ALP in skeletal mineralization, the precise function remains unclear. The initial site of mineralization (primary mineralization) normally occurs within the lumen of TNSALP-rich matrix vesicles (MVs) of growth cartilage, bone, and dentin. We investigated whether defective calcification in hypophosphatasia is due to a paucity and/or a functional failure of MVs secondary to TNSALP deficiency. Nondecalcified autopsy bone and growth plate cartilage from five patients with perinatal (lethal) hypophosphatasia were studied by nondecalcified light and electron microscopy to assess MV numbers, size, shape, and ultrastructure and whether hypophosphatasia MVs contain apatite-like mineral, as would be the case if these MVs retained their ability to concentrate calcium and phosphate internally despite a paucity of TNSALP in their investing membranes. We found that hypophosphatasia MVs are present in approximately normal numbers and distribution and that they are capable of initiating internal mineralization. There is retarded extravesicular crystal propagation. Thus, in hypophosphatasia the failure of bones to calcify appears to involve a block of the vectorial spread of mineral from initial nuclei within MVs, outwards, into the matrix. We conclude that hypophosphatasia MVs can concentrate calcium and phosphate internally despite a deficiency of TNSALP activity.     

Severe hypophosphatasia: characterization of fifteen novel mutations in the ALPL gene

Hypophosphatasia is an inherited disorder characterized by defective bone mineralization and deficiency of serum and tissue liver/bone/kidney alkaline phosphatase (L/B/K ALP) activity. We report the characterization of ALPL gene mutations in a series of 11 families from various origins affected by perinatal and infantile hypophosphatasia. Sixteen distinct mutations were found, fifteen of them not previously reported: M45V, G46R, 388-391delGTAA, 389delT, T131I, G145S, D172E, 662delG, G203A, R255L, 876-881delAGGGGA, 962delG, E294K, E435K, and A451T. This confirms that severe hypophosphatasia is due to a large spectrum of mutations in Caucasian populations.     

Complex arylsulfatase A alleles causing metachromatic leukodystrophy

Metachromatic leukodystrophy is a lysosomal storage disorder caused by the deficiency of arylsulfatase A. Sequencing of the arylsulfatase A genes of a patient affected with late infantile metachromatic leukodystrophy revealed that the patient is a compound heterozygote of two alleles carrying two deleterious mutation each. One allele bears a splice donor site mutation together with two polymorphisms and an additional missense mutation (Gly 122>Ser). The splice donor site mutation and the Gly 122>Ser substitution have been described recently but on different alleles. The other allele carries two missense mutations causing a Gly 154>Asp and a Pro 167>Arg substitution. When arylsulfatase A cDNAs carrying these mutations separately or in combination were transfected into baby hamster kidney cells expression of arylsulfatase A activity could not be detected. Linkage of mutations was verified by sequencing of the parental DNAs. Biosynthesis studies performed with the patients' fibroblasts show that the enzyme carrying both mutations is synthesized in almost normal amounts but is rapidly degraded in an early biosynthetic compartment. The occurence of two disease causing mutations on the same allele is a novel phenomenon in metachromatic leukodystrophy and as far as lysosomal storage diseases are concerned have so far only been described in Fabry disease and in the complex glucocerebrosidase alleles associated with Gaucher disease. © 1994 Wiley-Liss, Inc.     

Van Der Woude syndrome: variable penetrance of a novel mutation (p.Arg 84Gly) of the IRF6 gene in a Turkish family

Van der Woude syndrome (VWS) is an autosomal dominant disorder characterized by clefts of the lip and/or palate (CL+/-P), lip pits, bifid uvula and hypodontia. Mutations of the interferon regulatory factor 6 gene (IRF6) have been recently described in patients with VWS. The entire 9 exons of the IRF6 gene in two brothers of Turkish origin clinically diagnosed with Van der Woude syndrome and four healthy family members were screened for mutations using a newly established denaturing gradient gel electrophoresis (DGGE) method. A novel heterozygous mutation in exon 2 (DNA binding region) of the IRF6 gene, p.Arg84Gly, was found in both brothers with VWS and in their clinically asymptomatic mother. Our results suggest a dominant negative effect of the p.Arg84Gly mutation in the VWS of both patients. Non-penetrance of this mutation is suggested in the mother of the patients.     

Mutation screening of TRPM1, GRM6, NYX and CACNA1F genes in patients with congenital stationary night blindness

The aim of this study was to identify mutations in the TRPM1, GRM6, NYX and CACNA1F genes in patients with congenital stationary night blindness (CSNB). Twenty-four unrelated patients with CSNB were ascertained. Sanger sequencing was used to analyze the coding exons and adjacent intronic regions of TRPM1, GRM6, NYX and CACNA1F. Six mutations were identified in six unrelated patients, including five novel and one known. Of the six, three novel hemizygous mutations, c.92G>A (p.Cys31Tyr), c.149G>C (p.Ary50Pro), and c.[272T>A;1429G>C] (p.[Leu91Gln;Gly477Arg]), were found in NYX in three patients, respectively. A novel c.[1984_1986delCTC;3001G>A] (p.[Leu662del;Gly1001Arg]) mutation was detected in CACNA1F in one patient. One novel and one known heterozygous variation, c.1267T>C (p.Cys423Arg) and c.1537G>A (p.Val513Met), were detected in GRM6 in two patients, respectively. No variations were found in TRPM1. The results expand the mutation spectrum of NYX, CACNA1F and GRM6. They also suggest that NYX mutations are a common cause of CSNB.     

Mutations in MFSD8/CLN7 are a frequent cause of variant‐late infantile neuronal ceroid lipofuscinosis

The neuronal ceroid lipofuscinoses (NCL) are a group of genetically heterogeneous neurodegenerative disorders. The recent identification of the MFSD8/CLN7 gene in a variant‐late infantile form of NCL (v‐LINCL) in affected children from Turkey prompted us to examine the relative frequency of variants in this gene in Italian patients with v‐LINCL. We identified nine children harboring 11 different mutations in MFSD8/CLN7. Ten mutations were novel and included three nonsense (p.Arg35Stop, p.Glu381Stop, p.Arg482Stop), four missense (p.Met1Thr, p.Gly52Arg, p.Thr294Lys, p.Pro447Leu), two splice site mutations (c.863+3_4insT, c.863+1G>C), and a 17‐bp deletion predicting a frameshift and premature protein truncation (c.627_643del17/p.Met209IlefsX3). The clinical phenotype, which was similar to that of the Turkish v‐LINCL cases, was not influenced by type and location of the mutation nor the length of the predicted residual gene product. As well as identifying novel variants in MFSD8/CLN7, this study contributes to a better molecular characterization of Italian NCL cases, and will facilitate medical genetic counseling in such families. The existence of a subset of v‐LINCL cases without mutations in any of the known NCL genes suggests further genetic heterogeneity. © 2009 Wiley‐Liss, Inc.     

Correlations of genotype and phenotype in hypophosphatasia.

Hypophosphatasia, a rare inherited disorder characterized by defective bone mineralization, is highly variable in its clinical expression. The disease is due to various mutations in the tissue-non-specific alkaline phosphatase ( TNSALP ) gene. We report here the use of clinical data, site-directed mutagenesis and computer-assisted modelling to propose a classification of 32 TNSALP gene mutations found in 23 European patients, 17 affected with lethal hypophosphatasia and six with non-lethal hypophosphatasia. Transfection studies of the missense mutations found in non-lethal hypophosphatasia showed that six of them allowed significant residual in vitro enzymatic activity, suggesting that these mutations corresponded to moderate alleles. Each of the six patients with non-lethal hypophosphatasia carried at least one of these alleles. The three-dimensional model study showed that moderate mutations were not found in the active site, and that most of the severe missense mutations were localized in crucial domains such as the active site, the vicinity of the active site and homodimer interface. Some mutations appeared to be organized in clusters on the surface of the molecule that may represent possible candidates for regions interacting with the C-terminal end involved in glycosylphosphatidylinositol (GPI) attachment or with other dimers to form tetramers. Finally, our results show a good correlation between clinical forms of the disease, mutagenesis experiments and the three-dimensional structure study, and allowed us to clearly distinguish moderate alleles from severe alleles. They also confirm that the extremely high phenotypic heterogeneity observed in patients with hypophosphatasia was due mainly to variable residual enzymatic activities allowed by missense mutations found in the human TNSALP gene.     

Combined in vitro and in silico analyses of FGFR1 variants: genotype-phenotype study in idiopathic hypogonadotropic hypogonadism

Fibroblast growth factor receptor 1 (FGFR1) is an idiopathic hypogonadotropic hypogonadism (IHH)-associated gene, mutated in approximately 10% of the patients with this condition. Through targeted gene sequencing of 153 males with IHH and 100 healthy controls, we identified 10 mutations in FGFR1 from IHH patients with a frequency of 5.9% in the Chinese population of central China. These included nine missense mutations(NM_023110.2, p.Gly687Arg, p.Ala608Asp, p.Gly348Glu, p.Asn296Ser, p.Gly226Asp, p.Arg209Cys, p.Gly97Arg, p.Val71Met, p.Gly70Arg) and a splicing mutation c.1430 + 1G > T. in vitro and in silico analyses of FGFR1 variants were conducted to study the impact of the identified mutations. Our findings indicated that the splicing mutation dramatically affected premRNA processing, causing exon 10 and 6 nucleotides in the 3′ end of exon 9 to be completely skipped. Two variants (p.Gly687Arg and p.Ala608Asp) markedly impaired tyrosine kinase activity, while the other variants had limited impact on the mitogen-activated protein kinase (MAPK) signaling pathway. However, the functional impairment of the mutant receptors was not always consistent with the phenotypes, indicating that FGFR1 mutations might cause IHH in conjunction with other mutant genes. In this study, we expanded the knowledge on the mutation spectrum of FGFR1 in IHH patients and explored the genotype-phenotype relationship.     

Evidence of a founder effect for the tissue-nonspecific alkaline phosphatase (TNSALP) gene E174K mutation in hypophosphatasia patients

Hypophosphatasia is a rare inborn error of metabolism characterised by defective bone mineralisation caused by a deficiency of liver-, bone- or kidney-type alkaline phosphatase due to mutations in the tissue-nonspecific alkaline phosphatase (TNSALP) gene. The clinical expression of the disease is highly variable, ranging from stillbirth with a poorly mineralised skeleton to pathologic skeletal fractures which develop in late adulthood only. This clinical heterogeneity is due to the strong allelic heterogeneity in the TNSALP gene. We found that mutation E174K is the most frequent in Caucasian patients, and that it was carried by 31% of our patients with mild hypophosphatasia. Because the mutation was found in patients of various geographic origins, we investigated whether it had a unique origin or rather multiple origins due to recurrence of de novo mutations. Three intragenic polymorphisms, S93S, 472+12delG and V505A, were genotyped in patients carrying E174K and in normal unrelated individuals. Our results show that all the E174K mutations are carried by a common ancestral haplotype, also found at low frequency in normal and hypophosphatasia chromosomes. We conclude that the TNSALP gene E174K mutation is the result of a relatively ancient ancestral mutation that occurred on a single chromosome in the north of Western Europe and spread throughout the rest of Europe and into the New World as a result of human migration.     

High frequency of mutations in the PIK3CA gene helical and kinase coding regions in a group of Iranian patients with high-grade glioblastomas: five novel mutations

Glioblastoma multiform (GBM; World Health Organization (WHO) grade IV) and anaplastic astrocytomas (AA; WHO grade III) are highly aggressive and lethal astrocytic brain tumors. To detect cancer-specific somatic mutations in two hot-spot regions of PIK3CA gene, the helical and kinase domains (encoded by exons 9 and 20, respectively) in GBM and AA, the authors examined the respective sequences 31 paraffin-embedded samples (23 GBM and 8 AA). The samples were obtained from a group of Iranian patients affected with high-grade glioblastoma (HGG). The overall prevalence of PIK3CA mutations was 23% (7/31) for both tumor types (22% in GBM, and 25% in AA). Five mutations were detected in exon 20, p.Arg992Gln (c.2976G→A), p.Met1005Val (c.3014A→G), p.Ile1019→Val (c.3056A→G), p.Ser1008Cys (c.3024C→G), and p.Asn1044Asp (c.3130A→G), and one mutation in exon 9, p.Glu545Ala (c.1634A→C). Additionally exons 4-8 of P53 gene in four unrelated young patients, who showed no mutations in PIK3CA exons 9 and 20, were analyzed. Three mutations were identified: p.Pro72Ala (c.214C→G), g.11608G→T (homozygote splice mutation), and p.Thr170Thr (c.510G→A silent mutation). In conclusion, mutation detection in PIK3CA in patients with a high degree of malignancy and early age at diagnosis should be included in molecular diagnostic protocols, also with regard to possible upcoming therapies.     

Sensitivity of the denaturing gradient gel electrophoresis technique in detection of known mutations and novel Asian mutations in the CFTR gene

More than 500 mutations have been identified in the CFTR gene, making it an excellent system for testing mutation scanning techniques. To assess the sensitivity of denaturing gradient gel electrophoresis (DGGE), we collected a representative group of 202 CFTR mutations. All mutations analyzed were detected by scanning methods other than the DGGE approach evaluated in this study. DGGE analysis was performed on 24 of the 27 exons and their flanking splice site sequences. After optimization, 201 of the 202 control samples produced an altered migration pattern in the region in which an alteration occurred. The remaining sample was sequenced and found not to have the reported mutation. The ability of DGGE to identify novel mutations was evaluated in three Asian CF patients with four unknown CF alleles. Three novel Asian mutations were detected—K166E, L568X, and 3121-2 A→G (in homozygosity)—accounting for all CF alleles. These results indicate that an optimized DGGE scanning strategy is highly sensitive and specific and can detect 100% of mutations. Hum Mutat 9:136–147, l997. © 1997 Wiley-Liss, Inc.     

A DGGE system for comprehensive mutation screening of BRCA1 and BRCA2: application in a Dutch cancer clinic setting

Rapid and reliable identification of deleterious changes in the breast cancer genes BRCA1 and BRCA2 has become one of the major issues in most DNA services laboratories. To rapidly detect all possible changes within the coding and splice site determining sequences of the breast cancer genes, we established a semiautomated denaturing gradient gel electrophoresis (DGGE) mutation scanning system. All exons of both genes are covered by the DGGE scan, comprising 120 amplicons. We use a semiautomated approach, amplifying all individual amplicons with the same PCR program, after which the amplicons are pooled. DGGE is performed using three slightly different gel conditions. Validation was performed using DNA samples with known sequence variants in 107 of the 120 amplicons; all variants were detected. This DGGE mutation scanning, in combination with a PCR test for two Dutch founder deletions in BRCA1 was then applied in 431 families in which 52 deleterious changes and 70 unclassified variants were found. Fifteen unclassified variants were not reported before. The system was easily adopted by five other laboratories, where in another 3,593 families both exons 11 were analyzed by the protein truncation test (PTT) and the remaining exons by DGGE. In total, a deleterious change (nonsense, frameshift, splice-site mutation, or large deletion) was found in 661 families (16.4%), 462 in BRCA1 (11.5%), 197 in BRCA2 (4.9%), and in two index cases a deleterious change in both BRCA1 and BRCA2 was identified. Eleven deleterious changes in BRCA1 and 36 in BRCA2 had not been reported before. In conclusion, this DGGE mutation screening method for BRCA1 and BRCA2 is proven to be highly sensitive and is easy to adopt, which makes screening of large numbers of patients feasible. The results of screening of BRCA1 and BRCA2 in more than 4,000 families present a valuable overview of mutations in the Dutch population.     

High Frequency of Mutations in the PIK3CA Gene Helical and Kinase Coding Regions in a Group of Iranian Patients with High-Grade Glioblastomas: Five Novel Mutations

Abstract: Glioblastoma multiform (GBM; World Health Organization (WHO) grade IV) and anaplastic astrocytomas (AA; WHO grade III) are highly aggressive and lethal astrocytic brain tumors. To detect cancer-specific somatic mutations in two hot-spot regions of PIK3CA gene, the helical and kinase domains (encoded by exons 9 and 20, respectively) in GBM and AA, the authors examined the respective sequences 31 paraffin-embedded samples (23 GBM and 8 AA). The samples were obtained from a group of Iranian patients affected with high-grade glioblastoma (HGG). The overall prevalence of PIK3CA mutations was 23% (7/31) for both tumor types (22% in GBM, and 25% in AA). Five mutations were detected in exon 20, p.Arg992Gln (c.2976G→A), p.Met1005Val (c.3014A→G), p.Ile1019→Val (c.3056A→G), p.Ser1008Cys (c.3024C→G), and p.Asn1044Asp (c.3130A→G), and one mutation in exon 9, p.Glu545Ala (c.1634A→C). Additionally exons 4–8 of P53 gene in four unrelated young patients, who showed no mutations in PIK3CA exons 9 and 20, were analyzed. Three mutations were identified: p.Pro72Ala (c.214C→G), g.11608G→T (homozygote splice mutation), and p.Thr170Thr (c.510G→A silent mutation). In conclusion, mutation detection in PIK3CA in patients with a high degree of malignancy and early age at diagnosis should be included in molecular diagnostic protocols, also with regard to possible upcoming therapies.     

Perinatal hypophosphatasia: diagnosis and detection of heterozygote carriers within the family

We report on two families in which one or two children had a severe disorder of skeletal development detected by prenatal ultrasonography. The children died postnatally and showed typical radiological and biochemical findings of perinatal hypophosphatasia. Biochemical analysis revealed a low activity of alkaline phosphatase (AP) and a high value of pyridoxal-5-phosphate (PLP), one of its natural substrates. The screening for mutations of the tissue nonspecific alkaline phosphatase (TNSALP) gene showed homozygosity for a point mutation (G 317 --> D) in the two affected children of the first family. The affected child of the second family was homozygous for a nonsense mutation (R 411 --> X). Family screening revealed that the determination of AP and PLP is helpful for detection of heterozygotes. However, heterozygote children had values of AP in the lower normal range during phases of rapid growth. The determination of PLP proved to be more sensitive in these cases. It should be kept in mind that during the last trimester of gestation there is an increase in maternal AP activity and a normalization of PLP due to placental AP, which is not affected. Therefore, in the course of a prenatal diagnosis in an index case, paternal blood should be analyzed in parallel. For detailed genetic counseling and early prenatal diagnosis in following pregnancies, the possibility of mutation analysis should be used.     

Spectrum of ATP7B mutations and genotype–phenotype correlation in large‐scale Chinese patients with Wilson Disease

Wilson disease (WD), an inherited disorder associated with ATP7B gene, has a wide spectrum of genotypes and phenotypes. In this study, we developed a rapid multiplex PCR‐MassArray method for detecting 110 mutant alleles of interest, and used it to examine genomic DNA from 1222 patients and 110 healthy controls. In patients not found to have any mutation in the 110 selected alleles, PCR‐Sanger sequencing was used to examine the ATP7B gene. We identified 88 mutations, including 9 novel mutations. Our analyses revealed p.Arg778Leu, p.Arg919Gly and p.Thr935Met showed some correlations to phenotype. The p.Arg778Leu was related to younger onset age and lower levels of ceruloplasmin (Cp) and serum copper, while p.Arg919Gly and p.Thr935Met both indicated higher Cp levels. Besides, the p.Arg919Gly was related to neurological subtype, and p.Thr935Met showed significant difference in the percentage of combined neurological and visceral subtype. Moreover, for ATP7B mutations, the more severe impact on ATP7B protein was, the younger onset age and lower Cp level presented. The feasibility of presymptomatic DNA diagnosis and predicting clinical manifestation or severity of WD would be facilitated with identified mutations and genotype–phenotype correlation precisely revealed in the study.